Defining the Roles of Serine Palmitoyltransferase-Interacting Proteins in the Regulation of Sphingolipid Homeostasis

Sphingolipids are major structural components of the plasma membrane and endomembrane system. Research suggests that sphingolipids are involved with the formation of lipid microdomains, also known as lipid rafts, which may help to organize proteins within the membrane and may be important for membrane trafficking. Aside from their structural roles in membranes, sphingolipids and their metabolic products have been implicated in several cellular signaling responses like programmed cell death (PCD). Because of this, maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and development. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is the primary regulatory point for sphingolipid homeostasis. We have characterized two sets of Arabidopsis proteins that physically interact with and impact Arabidopsis SPT activity. The first set, the ssSPTs (Arabidopsis thaliana), have been shown to be essential and redundant. Modulation of AtssSPT expression was shown to alter SPT activity, LCB accumulation and sensitivity to the mycotoxin, Fumonisin B1 (FB1), in a way that is consistent with the AtssSPTs being activators of SPT. Alternatively, modulating expression of the other set of proteins, the AtORMs, was shown to alter SPT activity, LCB accumulation and sensitivity to FB1, consistent with them acting as SPT inhibitors. Both the AtssSPTs and the AtORMs appear to be limiting as transgenic up/down regulation of these genes leads to predictable changes to SPT activity. Interestingly, we also see changes in ceramide synthase activity with modulation of AtORM expression, suggesting a more complex regulatory role for these proteins and pointing towards coordinate regulation of SPT with downstream enzymes. Our research also demonstrates that SPT substrate specificity can be altered through point mutations in AtssSPT and AtLCB1, leading to the production of aberrant long-chain bases (LCBs). Alteration of SPT substrate specificity may function as another regulatory control point by altering SPT products through changes in the composition of SPT subunits. We also discuss a system utilizing a point mutation in AtLCB1 that can be used as a tool to better measure SPT activity in planta. Collectively our data point towards a complex and nuanced regulatory scheme for maintaining sphingolipid homeostasis in Arabidopsis thaliana. Advisor: Edgar Cahoo